Inherited epidermolysis bullosa (EB) is a mechanobullous disease, characterized by skin separation and the development of blisters following minor or insignificant trauma of the skin. Skin separation in the junctional form of EB (JEB) occurs within the lamina lucida of the cutaneous basement membrane zone and is associated with mutations in genes encoding the laminin 5 subunits, LAMAS, LAMBS or LAMC2. Current gene therapy approaches are not able to effectively rescue mutations in these genes and the resulting phenotypic manifestations. Therefore, the proposed research is focused on the development of an alternative, protein-based therapeutic approach that can be used for the management and treatment of local blisters that usually arise on patients with a non-lethal form of JEB at the sites of mechanical stress (fingers, elbows, knees, etc). The current project is based on the preliminary data demonstrating that a recombinant chains of human laminin 5 can be produced and purified in vitro, and being delivered into keratinocytes isolated from JEB patients, can restore the assembly of mature laminin 5. The first aim of this project is to optimize large-scale production and purification of recombinant beta3 and gamma 2 chains of human laminin 5 as they are often mutated in JEB. It is also planned to produce and purify recombinant mouse laminin 5 chains that will be used on pre-clinical JEB animal models. In the second aim it is planned to test the ability of these recombinant chains to substitute mutant laminin 5 subunits in vitro, on keratinocytes isolated from JEB patients and from JEB mice. It is also planned to assess the functionality of these recombinant proteins by various in vitro assays. In the third specific aim it is planned to evaluate the functionality of recombinant proteins and the applicability of the suggested protein-based strategy on human skin reconstructs and on LAMB3 and LAMC2 (LamB3IAP and Lamc2-/-) deficient mice. Overall, the proposed study will evaluate the feasibility of protein therapy for the management and treatment of local blisters in JEB and potentially can result in the development of new therapeutics that allow blister prevention and alleviation of painful symptoms associated with JEB.